bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2023–12–24
fifty-five papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Mitochondrial quality control pathways sense mitochondrial protein import.
  2. Muscle immune cells protect mitochondrial organelles during exercise.
  3. MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).
  4. SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.
  5. Case report: Mutations in DNAJC30 causing autosomal recessive Leber hereditary optic neuropathy are common amongst Eastern European individuals.
  6. Layered mechanisms regulating the human mitochondrial NAD+ transporter SLC25A51.
  7. Mitochondrial quality control via organelle and protein degradation.
  8. Mitochondrial network reorganization and transient expansion during oligodendrocyte generation.
  9. Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network.
  10. FBXL4 mutation-caused mitochondrial DNA depletion syndrome is driven by BNIP3/BNIP3L-dependent excessive mitophagy.
  11. Mitochondrial Quality Control via Mitochondrial Unfolded Protein Response (mtUPR) in Ageing and Neurodegenerative Diseases.
  12. PNPLA3-associated mitochondrial dysfunction.
  13. Mitochondrial fatty acid synthesis is an emergent central regulator of mammalian oxidative metabolism.
  14. Human-specific translational control of neuronal mitochondria and excitability.
  15. Gene dosage adaptations to mtDNA depletion and mitochondrial protein stress in budding yeast.
  16. Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.
  17. DNAJC30 Gene Variants Are a Frequent Cause of a Rare Disease: Leber Hereditary Optic Neuropathy in Polish Patients.
  18. Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).
  19. GeneMAP: A discovery platform for metabolic gene function.
  20. Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology.
  21. Leber hereditary optic neuropathy gene therapy.
  22. Challenges in Genetic Diagnosis of Mitochondrial Diseases: What Can Functional Genomics' Studies Do?
  23. Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis.
  24. Schizosaccharomyces pombe as a fundamental model for research on mitochondrial gene expression: Progress, achievements and outlooks.
  25. Impact of platelet activation on the release of cell-free mitochondria and circulating mitochondrial DNA.
  26. Glucose-mediated mitochondrial reprogramming by cholesterol export at TM4SF5-enriched mitochondria-lysosome contact sites.
  27. Metabolic Alterations in NADSYN1-Deficient Cells.
  28. One-pot, degradable, silica nanocarriers with encapsulated oligonucleotides for mitochondrial specific targeting.
  29. Quantitative Analysis of Mitochondrial DNA Heteroplasmy in Urinary Podocytes of Myoclonus Epilepsy With Ragged-Red Fibers Syndrome.
  30. Complex II ambiguities-FADH2 in the electron transfer system.
  31. Mitochondrial transplantation attenuates oligomeric amyloid-beta-induced mitochondrial dysfunction and tight junction protein destruction in retinal pigment epithelium.
  32. Magnetic resonance spectroscopy in MELAS syndrome: correlation with CSF and plasma metabolite levels and change after glutamine supplementation.
  33. Cyclohexylalanine-Containing α-Helical Amphipathic Peptide Targets Cardiolipin, Rescuing Mitochondrial Dysfunction in Kidney Injury.
  34. Generation of inducible mitophagy mice.
  35. ClinGen guidance for use of the PP1/BS4 co-segregation and PP4 phenotype specificity criteria for sequence variant pathogenicity classification.
  36. Disruption of Mitophagy Flux through the PARL-PINK1 Pathway by CHCHD10 Mutations or CHCHD10 Depletion.
  37. Ago2 Protects Against Diabetic Cardiomyopathy by Activating Mitochondrial Gene Translation.
  38. Mitochondrial homeostasis regulation: A promising therapeutic target for Parkinson's disease.
  39. Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.
  40. An integrative framework for clinical diagnosis and knowledge discovery from exome sequencing data.
  41. Automatically pre-screening patients for the rare disease aromatic l-amino acid decarboxylase deficiency using knowledge engineering, natural language processing, and machine learning on a large EHR population.
  42. Translation Fidelity and Respiration Deficits in CLPP-Deficient Tissues: Mechanistic Insights from Mitochondrial Complexome Profiling.
  43. Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL): Report of a new family and a novel DARS2 mutation.
  44. Dissecting organelle interdependence.
  45. Benchmarking splice variant prediction algorithms using massively parallel splicing assays.
  46. A comprehensive study of mutation and phenotypic heterogeneity of childhood mitochondrial leukodystrophies.
  47. An overview of ATP synthase, inhibitors, and their toxicity.
  48. Gene therapy for Parkinson's disease models.
  49. Human retinal organoids with an OPA1 mutation are defective in retinal ganglion cell differentiation and function.
  50. Neurological gait assessment.
  51. Structural insights into N-terminal methionine cleavage by the human mitochondrial methionine aminopeptidase, MetAP1D.
  52. REAP+: A single preparation for rapid isolation of nuclei, cytoplasm, and mitochondria.
  53. Need for speed: Human fast-twitch mitochondria favor power over efficiency.
  54. Real-Time Monitoring of Mitochondrial Damage Using Conjugated Oligoelectrolytes.
  55. Streamlining spatial omics data analysis with Pysodb.
  1. Trends Endocrinol Metab. 2023 Dec 15. pii: S1043-2760(23)00243-6. [Epub ahead of print]
    Mitochondrial quality control pathways sense mitochondrial protein import.
    Laurie P Lee-Glover, Timothy E Shutt.
      Mitochondrial quality control (MQC) mechanisms are required to maintain a functional proteome, which enables mitochondria to perform a myriad of important cellular functions from oxidative phosphorylation to numerous other metabolic pathways. Mitochondrial protein homeostasis begins with the import of over 1000 nuclear-encoded mitochondrial proteins and the synthesis of 13 mitochondrial DNA-encoded proteins. A network of chaperones and proteases helps to fold new proteins and degrade unnecessary, damaged, or misfolded proteins, whereas more extensive damage can be removed by mitochondrial-derived vesicles (MDVs) or mitochondrial autophagy (mitophagy). Here, focusing on mechanisms in mammalian cells, we review the importance of mitochondrial protein import as a sentinel of mitochondrial function that activates multiple MQC mechanisms when impaired.
    Keywords:  mitochondria; mitochondrial protein import; mitochondrial quality control; mitochondrial unfolded protein response; mitochondrial-derived vesicles; mitophagy
    DOI:  https://doi.org/10.1016/j.tem.2023.11.004
  2. Nature. 2024 Jan;625(7993): 35-36
    Muscle immune cells protect mitochondrial organelles during exercise.
    Gerald Coulis, S Armando Villalta.
      
    Keywords:  Cell biology; Immunology; Physiology
    DOI:  https://doi.org/10.1038/d41586-023-03972-w
  3. Int J Mol Sci. 2023 Dec 06. pii: 17186. [Epub ahead of print]24(24):
    MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).
    Lu Liu, Jiahao Yang, Yoshinori Otani, Takahiro Shiga, Akihiro Yamaguchi, Yasuaki Oda, Miho Hattori, Tsukimi Goto, Shuichi Ishibashi, Yuki Kawashima-Sonoyama, Takaya Ishihara, Yumi Matsuzaki, Wado Akamatsu, Masashi Fujitani, Takeshi Taketani.
      Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.
    Keywords:  MELAS; mesenchymal stem cells (MSCs); mitochondrial transfer; rapidly expanding clones (RECs)
    DOI:  https://doi.org/10.3390/ijms242417186
  4. bioRxiv. 2023 Dec 08. pii: 2023.12.06.570371. [Epub ahead of print]
    SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology.
    Taolin Yuan, Surinder Kumar, Mary Skinner, Ryan Victor-Joseph, Majd Abuaita, Jaap Keijer, Jessica Zhang, Thaddeus J Kunkel, Yanghan Liu, Elyse M Petrunak, Thomas L Saunders, Andrew P Lieberman, Jeanne A Stuckey, Nouri Neamati, Fathiya Al-Murshedi, Majid Alfadhel, Johannes N Spelbrink, Richard Rodenburg, Vincent C J de Boer, David B Lombard.
      SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.
    DOI:  https://doi.org/10.1101/2023.12.06.570371
  5. Front Neurol. 2023 ;14 1292320
    Case report: Mutations in DNAJC30 causing autosomal recessive Leber hereditary optic neuropathy are common amongst Eastern European individuals.
    Toby Charles Major, Eszter Sara Arany, Katherine Schon, Magdolna Simo, Veronika Karcagi, Jelle van den Ameele, Patrick Yu Wai Man, Patrick F Chinnery, Catarina Olimpio, Rita Horvath.
       Background: Leber Hereditary Optic Neuropathy (LHON) is the most common inherited mitochondrial disease characterized by bilateral, painless, subacute visual loss with a peak age of onset in the second to third decade. Historically, LHON was thought to be exclusively maternally inherited due to mutations in mitochondrial DNA (mtDNA); however, recent studies have identified an autosomal recessive form of LHON (arLHON) caused by point mutations in the nuclear gene, DNAJC30.
    Case Presentations: In this study, we report the cases of three Eastern European individuals presenting with bilateral painless visual loss, one of whom was also exhibiting motor symptoms. After a several-year-long diagnostic journey, all three patients were found to carry the homozygous c.152A>G (p.Tyr51Cys) mutation in DNAJC30. This has been identified as the most common arLHON pathogenic variant and has been shown to exhibit a significant founder effect amongst Eastern European individuals.
    Conclusion: This finding adds to the growing cohort of patients with arLHON and demonstrates the importance of DNAJC30 screening in patients with molecularly undiagnosed LHON, particularly in Eastern European individuals. It is of heightened translational significance as patients diagnosed with arLHON exhibit a better prognosis and response to therapeutic treatment with the co-enzyme Q10 analog idebenone.
    Keywords:  DNA-J heat shock protein family (Hsp40) member C30 (DNAJC30); Leber hereditary optic neuropathy (LHON); autosomal recessive LHON (arLHON); c.152A>G (p.Tyr51Cys); idebenone; mitochondrial LHON (mtLHON); recessive optic neuropathy
    DOI:  https://doi.org/10.3389/fneur.2023.1292320
  6. Biochem Soc Trans. 2023 Dec 20. 51(6): 1989-2004
    Layered mechanisms regulating the human mitochondrial NAD+ transporter SLC25A51.
    Shivansh Goyal, Xiaolu A Cambronne.
      SLC25A51 is the primary mitochondrial NAD+ transporter in humans and controls many local reactions by mediating the influx of oxidized NAD+. Intriguingly, SLC25A51 lacks several key features compared with other members in the mitochondrial carrier family, thus its molecular mechanism has been unclear. A deeper understanding would shed light on the control of cellular respiration, the citric acid cycle, and free NAD+ concentrations in mammalian mitochondria. This review discusses recent insights into the transport mechanism of SLC25A51, and in the process highlights a multitiered regulation that governs NAD+ transport. The aspects regulating SLC25A51 import activity can be categorized as contributions from (1) structural characteristics of the transporter itself, (2) its microenvironment, and (3) distinctive properties of the transported ligand. These unique mechanisms further evoke compelling new ideas for modulating the activity of this transporter, as well as new mechanistic models for the mitochondrial carrier family.
    Keywords:  MCART1; NAD; SLC25A51; mitochondrial carrier family; mitochondrial transport
    DOI:  https://doi.org/10.1042/BST20220318
  7. J Biochem. 2023 Dec 15. pii: mvad106. [Epub ahead of print]
    Mitochondrial quality control via organelle and protein degradation.
    Koji Yamano, Hiroki Kinefuchi, Waka Kojima.
      Mitochondria are essential eukaryotic organelles that produce ATP as well as synthesize various macromolecules. They also participate in signaling pathways such as the innate immune response and apoptosis. These diverse functions are performed by >1000 different mitochondrial proteins. Although mitochondria are continuously exposed to potentially damaging conditions such as reactive oxygen species, proteases/peptidases localized in different mitochondrial sub-compartments, termed mitoproteases, maintain mitochondrial quality and integrity. In addition to processing incoming precursors and degrading damaged proteins, mitoproteases also regulate metabolic reactions, mitochondrial protein half-lives, and gene transcription. Impaired mitoprotease function is associated with various pathologies. In this review, we highlight recent advances in our understanding of mitochondrial quality control regulated by autophagy, ubiquitin-proteasomes, and mitoproteases.
    Keywords:  autophagy; mitophagy; peptidase; protease; ubiquitin
    DOI:  https://doi.org/10.1093/jb/mvad106
  8. bioRxiv. 2023 Dec 05. pii: 2023.12.05.570104. [Epub ahead of print]
    Mitochondrial network reorganization and transient expansion during oligodendrocyte generation.
    Xhoela Bame, Robert A Hill.
      Oligodendrocyte precursor cells (OPCs) give rise to myelinating oligodendrocytes of the central nervous system. This process persists throughout life and is essential for recovery from neurodegeneration. To better understand the cellular checkpoints that occur during oligodendrogenesis, we determined the mitochondrial distribution and morphometrics across the oligodendrocyte lineage in mouse and human cerebral cortex. During oligodendrocyte generation, mitochondrial content expanded concurrently with a change in subcellular partitioning towards the distal processes. These changes were followed by an abrupt loss of mitochondria in the oligodendrocyte processes and myelin, coinciding with sheath compaction. This reorganization and extensive expansion and depletion took 3 days. Oligodendrocyte mitochondria were stationary over days while OPC mitochondrial motility was modulated by animal arousal state within minutes. Aged OPCs also displayed decreased mitochondrial size, content, and motility. Thus, mitochondrial dynamics are linked to oligodendrocyte generation, dynamically modified by their local microenvironment, and altered in the aging brain.
    DOI:  https://doi.org/10.1101/2023.12.05.570104
  9. Nat Cell Biol. 2023 Dec 21.
    Quantitative subcellular reconstruction reveals a lipid mediated inter-organelle biogenesis network.
    Richard G Lee, Danielle L Rudler, Samuel A Raven, Liuyu Peng, Anaëlle Chopin, Edward S X Moh, Tim McCubbin, Stefan J Siira, Samuel V Fagan, Nicholas J DeBono, Maike Stentenbach, Jasmin Browne, Filip F Rackham, Ji Li, Kaylene J Simpson, Esteban Marcellin, Nicolle H Packer, Gavin E Reid, Benjamin S Padman, Oliver Rackham, Aleksandra Filipovska.
      The structures and functions of organelles in cells depend on each other but have not been systematically explored. We established stable knockout cell lines of peroxisomal, Golgi and endoplasmic reticulum genes identified in a whole-genome CRISPR knockout screen for inducers of mitochondrial biogenesis stress, showing that defects in peroxisome, Golgi and endoplasmic reticulum metabolism disrupt mitochondrial structure and function. Our quantitative total-organelle profiling approach for focussed ion beam scanning electron microscopy revealed in unprecedented detail that specific organelle dysfunctions precipitate multi-organelle biogenesis defects, impair mitochondrial morphology and reduce respiration. Multi-omics profiling showed a unified proteome response and global shifts in lipid and glycoprotein homeostasis that are elicited when organelle biogenesis is compromised, and that the resulting mitochondrial dysfunction can be rescued with precursors for ether-glycerophospholipid metabolic pathways. This work defines metabolic and morphological interactions between organelles and how their perturbation can cause disease.
    DOI:  https://doi.org/10.1038/s41556-023-01297-4
  10. Trends Mol Med. 2023 Dec 19. pii: S1471-4914(23)00279-4. [Epub ahead of print]
    FBXL4 mutation-caused mitochondrial DNA depletion syndrome is driven by BNIP3/BNIP3L-dependent excessive mitophagy.
    Kun Gao, Xiayun Xu, Chenji Wang.
      Encephalomyopathic mitochondrial DNA (mtDNA) depletion syndrome 13 (MTDPS13) is an autosomal recessive disorder arising from biallelic F-box and leucine-rich repeat (LRR) protein 4 (FBXL4) gene mutations. Recent advances have shown that excessive BCL2 interacting protein 3 (BNIP3)/ BCL2 interacting protein 3 like (BNIP3L)-dependent mitophagy underlies the molecular pathogenesis of MTDPS13. Here, we provide an overview of these groundbreaking findings and discuss potential therapeutic strategies for this fatal disease.
    Keywords:  BNIP3/BNIP3L; FBXL4; MTDPS13; mitochondria; mitophagy; ubiquitination
    DOI:  https://doi.org/10.1016/j.molmed.2023.11.017
  11. Biomolecules. 2023 Dec 13. pii: 1789. [Epub ahead of print]13(12):
    Mitochondrial Quality Control via Mitochondrial Unfolded Protein Response (mtUPR) in Ageing and Neurodegenerative Diseases.
    Paula Cilleros-Holgado, David Gómez-Fernández, Rocío Piñero-Pérez, Jose Manuel Romero-Domínguez, Diana Reche-López, Alejandra López-Cabrera, Mónica Álvarez-Córdoba, Manuel Munuera-Cabeza, Marta Talaverón-Rey, Alejandra Suárez-Carrillo, Ana Romero-González, Jose Antonio Sánchez-Alcázar.
      Mitochondria play a key role in cellular functions, including energy production and oxidative stress regulation. For this reason, maintaining mitochondrial homeostasis and proteostasis (homeostasis of the proteome) is essential for cellular health. Therefore, there are different mitochondrial quality control mechanisms, such as mitochondrial biogenesis, mitochondrial dynamics, mitochondrial-derived vesicles (MDVs), mitophagy, or mitochondrial unfolded protein response (mtUPR). The last item is a stress response that occurs when stress is present within mitochondria and, especially, when the accumulation of unfolded and misfolded proteins in the mitochondrial matrix surpasses the folding capacity of the mitochondrion. In response to this, molecular chaperones and proteases as well as the mitochondrial antioxidant system are activated to restore mitochondrial proteostasis and cellular function. In disease contexts, mtUPR modulation holds therapeutic potential by mitigating mitochondrial dysfunction. In particular, in the case of neurodegenerative diseases, such as primary mitochondrial diseases, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic Lateral Sclerosis (ALS), or Friedreich's Ataxia (FA), there is a wealth of evidence demonstrating that the modulation of mtUPR helps to reduce neurodegeneration and its associated symptoms in various cellular and animal models. These findings underscore mtUPR's role as a promising therapeutic target in combating these devastating disorders.
    Keywords:  ageing; mitochondria; mitochondrial biogenesis; mitochondrial diseases; mitochondrial dynamics; mitochondrial quality control mechanisms; mitochondrial unfolded protein response (mtUPR); mitophagy; neurodegenerative diseases; therapeutic target
    DOI:  https://doi.org/10.3390/biom13121789
  12. Nat Genet. 2023 Dec 18.
    PNPLA3-associated mitochondrial dysfunction.
    Kyle Vogan.
      
    DOI:  https://doi.org/10.1038/s41588-023-01636-z
  13. Cell Metab. 2023 Dec 14. pii: S1550-4131(23)00449-7. [Epub ahead of print]
    Mitochondrial fatty acid synthesis is an emergent central regulator of mammalian oxidative metabolism.
    Riley J Wedan, Jacob Z Longenecker, Sara M Nowinski.
      Contrary to their well-known functions in nutrient breakdown, mitochondria are also important biosynthetic hubs and express an evolutionarily conserved mitochondrial fatty acid synthesis (mtFAS) pathway. mtFAS builds lipoic acid and longer saturated fatty acids, but its exact products, their ultimate destination in cells, and the cellular significance of the pathway are all active research questions. Moreover, why mitochondria need mtFAS despite their well-defined ability to import fatty acids is still unclear. The identification of patients with inborn errors of metabolism in mtFAS genes has sparked fresh research interest in the pathway. New mammalian models have provided insights into how mtFAS coordinates many aspects of oxidative mitochondrial metabolism and raise questions about its role in diseases such as obesity, diabetes, and heart failure. In this review, we discuss the products of mtFAS, their function, and the consequences of mtFAS impairment across models and in metabolic disease.
    Keywords:  fatty acids; inborn errors of metabolism; lipid metabolism; lipids; mitochondria; mitochondrial fatty acid synthesis; mouse models; mtFAS
    DOI:  https://doi.org/10.1016/j.cmet.2023.11.017
  14. Neuron. 2023 Dec 20. pii: S0896-6273(23)00836-X. [Epub ahead of print]111(24): 3901-3903
    Human-specific translational control of neuronal mitochondria and excitability.
    Alicia R Lane, Meghan E Wynne, Victor Faundez.
      How are human-specific brain bioenergetics and excitability connected? In this Neuron issue, Shen et al.1 reveal a human-specific interaction between RACK1 mRNA and FMRP. Reducing RACK1 mimics FMRP-dependent excitability and mitochondrial phenotypes, which can be reversed with mitochondrial-protective drugs. These findings suggest that FMRP-mediated translation adapts mitochondria to excitability energy demands.
    DOI:  https://doi.org/10.1016/j.neuron.2023.10.031
  15. G3 (Bethesda). 2023 Dec 21. pii: jkad272. [Epub ahead of print]
    Gene dosage adaptations to mtDNA depletion and mitochondrial protein stress in budding yeast.
    Joshua T McNamara, Jin Zhu, Yuhao Wang, Rong Li.
      Mitochondria contain a local genome (mtDNA) comprising a small number of genes necessary for respiration, mitochondrial transcription and translation, and other vital functions. Various stressors can destabilize mtDNA leading to mtDNA loss. While some cells can survive mtDNA loss, they exhibit various deficiencies. Here, we investigated the impact of proteotoxicity on mitochondrial function by inducing mitochondrial unfolded protein stress in budding yeast. This led to rapid mtDNA loss, but aerobic conditioning imparted transient resistance to mitochondrial protein stress. We present a quantitative model of mtDNA loss in a growing cell population and measure its parameters. To identify genetic adaptations to mtDNA depletion, we performed a genome-wide screen for gene dosage increases that affect the growth of cells lacking mtDNA. The screen revealed a set of dosage suppressors that alleviate the growth impairment in mtDNA-deficient cells. Additionally, we show that these suppressors of mtDNA stress both bolster cell proliferation and prevent mtDNA loss during mitochondrial protein stress.
    Keywords:  copy number variation; functional genomics; mitochondria; mtDNA; protein aggregates; proteostasis; respiration; stress resistance
    DOI:  https://doi.org/10.1093/g3journal/jkad272
  16. Nature. 2023 Dec 20.
    Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.
    Chrysanthi Moschandrea, Vangelis Kondylis, Ioannis Evangelakos, Marija Herholz, Farina Schneider, Christina Schmidt, Ming Yang, Sandra Ehret, Markus Heine, Michelle Y Jaeckstein, Karolina Szczepanowska, Robin Schwarzer, Linda Baumann, Theresa Bock, Efterpi Nikitopoulou, Susanne Brodesser, Marcus Krüger, Christian Frezza, Joerg Heeren, Aleksandra Trifunovic, Manolis Pasparakis.
      Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.
    DOI:  https://doi.org/10.1038/s41586-023-06857-0
  17. Int J Mol Sci. 2023 Dec 15. pii: 17496. [Epub ahead of print]24(24):
    DNAJC30 Gene Variants Are a Frequent Cause of a Rare Disease: Leber Hereditary Optic Neuropathy in Polish Patients.
    Anna Skorczyk-Werner, Katarzyna Tońska, Aleksandra Maciejczuk, Katarzyna Nowomiejska, Magdalena Korwin, Monika Ołdak, Anna Wawrocka, Maciej R Krawczyński.
      Leber hereditary optic neuropathy (LHON) is a rare disorder causing a sudden painless loss of visual acuity in one or both eyes, affecting young males in their second to third decade of life. The molecular background of the LHON is up to 90%, genetically defined by a point mutation in mitochondrial DNA. Recently, an autosomal recessive form of LHON (LHONAR1, arLHON) has been discovered, caused by biallelic variants in the DNAJC30 gene. This study provides the results of the DNAJC30 gene analysis in a large group of 46 Polish patients diagnosed with LHON, together with the clinical characterization of the disease. The c.152A>G (p.Tyr51Cys) substitution in the DNAJC30 gene was detected in all the patients as homozygote or compound heterozygote. Moreover, we identified one novel variant, c.293A>G, p.(Tyr98Cys), as well as two ultra-rare DNAJC30 variants: c.293A>C, p.(Tyr98Ser), identified to date only in one individual affected with LHONAR1, and c.130_131delTC (p.Ser44ValfsTer8), previously described only in two patients with Leigh syndrome. The patients presented here represent the largest group of subjects with DNAJC30 gene mutations described to date. Based on our data, the autosomal recessive form of LHON caused by DNAJC30 gene mutations is more frequent than the mitochondrial form in Polish patients. The results of our study suggest that Sanger sequencing of the single-exon DNAJC30 gene should be a method of choice applied to identify a molecular background of clinically confirmed LHON in Polish patients. This approach will help to reduce the costs of molecular testing.
    Keywords:  DNAJC30; Leber hereditary optic neuropathy (LHON); arLHON); autosomal recessive LHON (LHONAR; mtDNA (mitochondrial DNA)
    DOI:  https://doi.org/10.3390/ijms242417496
  18. Nucleic Acids Res. 2023 Dec 19. pii: gkad1197. [Epub ahead of print]
    Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).
    Raja Norazireen Raja Ahmad, Long-Teng Zhang, Rikuri Morita, Haruna Tani, Yong Wu, Takeshi Chujo, Akiko Ogawa, Ryuhei Harada, Yasuteru Shigeta, Kazuhito Tomizawa, Fan-Yan Wei.
      MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.
    DOI:  https://doi.org/10.1093/nar/gkad1197
  19. bioRxiv. 2023 Dec 08. pii: 2023.12.07.570588. [Epub ahead of print]
    GeneMAP: A discovery platform for metabolic gene function.
    Artem Khan, Gokhan Unlu, Phillip Lin, Yuyang Liu, Ece Kilic, Timothy C Kenny, Kıvanç Birsoy, Eric R Gamazon.
      Organisms maintain metabolic homeostasis through the combined functions of small molecule transporters and enzymes. While many of the metabolic components have been well-established, a substantial number remains without identified physiological substrates. To bridge this gap, we have leveraged large-scale plasma metabolome genome-wide association studies (GWAS) to develop a multiomic Gene-Metabolite Associations Prediction (GeneMAP) discovery platform. GeneMAP can generate accurate predictions, even pinpointing genes that are distant from the variants implicated by GWAS. In particular, our work identified SLC25A48 as a genetic determinant of plasma choline levels. Mechanistically, SLC25A48 loss strongly impairs mitochondrial choline import and synthesis of its downstream metabolite, betaine. Rare variant testing and polygenic risk score analyses have elucidated choline-relevant phenomic consequences of SLC25A48 dysfunction. Altogether, our study proposes SLC25A48 as a mitochondrial choline transporter and provides a discovery platform for metabolic gene function.
    DOI:  https://doi.org/10.1101/2023.12.07.570588
  20. bioRxiv. 2023 Dec 10. pii: 2023.12.09.569632. [Epub ahead of print]
    Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology.
    Sunita Maharjan, Howard Gamper, Yuka Yamaki, Robert Y Henley, Nan-Sheng Li, Takeo Suzuki, Tsutomu Suzuki, Joseph A Piccirilli, Meni Wanunu, Erin Seifert, Douglas C Wallace, Ya-Ming Hou.
      Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m 1 G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.
    DOI:  https://doi.org/10.1101/2023.12.09.569632
  21. Curr Opin Ophthalmol. 2023 Dec 21.
    Leber hereditary optic neuropathy gene therapy.
    Byron L Lam.
       PURPOSE OF REVIEW: To discuss relevant clinical outcomes, challenges, and future opportunities of gene therapy in Leber hereditary optic neuropathy (LHON).
    RECENT FINDINGS: Results of G11778A LHON Phase 3 randomized clinical trials with unilateral intravitreal rAAV2/2-ND4 allotopic gene therapy show good safety and unexpected bilateral partial improvements of BCVA (best-corrected visual acuity) with mean logMAR BCVA improvements of up to near ∼0.3 logMAR (3 lines) in the treated eyes and ∼0.25 logMAR (2.5 lines) in the sham-treated or placebo-treated fellow eyes. Final mean BCVA levels after gene therapy were in the range of ∼1.3 logMAR (20/400) bilaterally.
    SUMMARY: Bilateral partial improvement with unilateral LHON gene therapy was unanticipated and may be due to treatment efficacy, natural history, learning effect, and other mediators. The overall efficacy is limited given the final BCVA levels. The sequential progressive visual loss and varied occurrence of spontaneous partial improvement in LHON confound trial results. Future clinical trials with randomization of patients to a group not receiving gene therapy in either eye would help to assess treatment effect. Promising future LHON gene therapy strategies include mitochondrially-targeted-sequence adeno-associated virus ('MTS-AAV') for direct delivery of the wild-type mitochondrial DNA into the mitochondria and CRISPR-free, RNA-free mitochondrial base editing systems. Signs of anatomical optic nerve damage and objective retinal ganglion cell dysfunction are evident in the asymptomatic eyes of LHON patients experiencing unilateral visual loss, indicating the therapeutic window is narrowing before onset of visual symptoms. Future treatment strategies utilizing mitochondrial base editing in LHON carriers without optic neuropathy holds the promise of a more advantageous approach to achieve optimal visual outcome by reducing disease penetrance and mitigating retinal ganglion cell loss when optic neuropathy develops.
    DOI:  https://doi.org/10.1097/ICU.0000000000001028
  22. Endocr Metab Immune Disord Drug Targets. 2023 Dec 18.
    Challenges in Genetic Diagnosis of Mitochondrial Diseases: What Can Functional Genomics' Studies Do?
    Marta Simões, Maria João Santos, Sara Martins, Maria do Carmo Macário, João Durães, Luísa Diogo, João Paulo Oliveira, José Carlos Ferreira, Manuela Grazina.
       INTRODUCTION: Mitochondrial oxidative phosphorylation (OXPHOS) diseases are challenging both from clinical and therapeutic perspectives. The advent of next-generation sequencing (NGS) boosted the discovery of new genetic defects affecting OXPHOS, with pathogenic variants identified in >350 genes to date [1]. However, in many patients, novel variants of unknown clinical significance are found. Subsequent functional studies may clarify its pathogenic consequences and modify the variant's classification, establishing a genetic diagnosis [2, 3].
    METHODS: Analysis of data obtained from patients (P1-P5) with novel genetic causes and functional genomics' studies performed, namely OXPHOS respiratory/glycolytic rates (Seahorse XF), enzymatic activity and assembly (BN-page), protein levels (SDS-WB), single muscle fiber assay, NGS and bioinformatics.
    RESULTS/CASE REPORT: P1-Leigh syndrome (40y, male); Complex IV activity deficiency (full assembly absent), homozygous deletion (c.-11_13del, SURF1), not detected by NGS[2]. P2- Epileptic encephalopathy (8y, male); homozygous c.882-1G>A, FASTKD2; OXPHOS decrease; reduced FASTKD2 expression and abnormal respiratory/glycolytic rates. P3-Cardiomyopathy/ nephropathy (39y, male); c.29G>C, FASTKD2; OXPHOS decrease; reduced FASTKD2 levels. P4-CPEO (62y, female); multiple OXPHOS deficiency; mtDNA alterations (m.7486G>A, MTTS1; 4,977bp del); higher levels of mutant mtDNA alterations in COX-deficient fibers [3]. P5- Polyneuropathy (15y, female); heterozygous c.1437C>A, POLG; combined def. or normal OXPHOS activity/respiratory capacity (tissue variable), raised CI assembly; normal POLG levels. Also, proteins' expression levels were reduced (P1-4), confirming pathogenicity. In P5, data do not support pathogenicity.
    CONCLUSION: If specific functional results are similar to controls, one might inquire about the pathogenicity of the studied variant and more genetic or bioinformatics analyses and family investigations are needed. There are also limitations of NGS in mutation detection that Sanger sequencing can overcome (P1). When performed first, the OXPHOS activity may guide to genetic screening or interpretation, concordant to later assembly results. All cases were solved and data may be crucial for genetic counseling.
    Keywords:  OXPHOS; functional genomics; laboratorial diagnosis; methodology; mitochondrial cytopathies; rare diseases
    DOI:  https://doi.org/10.2174/0118715303273290231211062420
  23. Nat Commun. 2023 Dec 20. 14(1): 8474
    Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis.
    Cecilia Patitucci, Juan Diego Hernández-Camacho, Elodie Vimont, Sonny Yde, Thomas Cokelaer, Thibault Chaze, Quentin Giai Gianetto, Mariette Matondo, Anastasia Gazi, Ivan Nemazanyy, David A Stroud, Daniella H Hock, Erminia Donnarumma, Timothy Wai.
      Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.
    DOI:  https://doi.org/10.1038/s41467-023-44143-9
  24. IUBMB Life. 2023 Dec 20.
    Schizosaccharomyces pombe as a fundamental model for research on mitochondrial gene expression: Progress, achievements and outlooks.
    Nhu Dinh, Nathalie Bonnefoy.
      Schizosaccharomyces pombe (fission yeast) is an attractive model for mitochondrial research. The organism resembles human cells in terms of mitochondrial inheritance, mitochondrial transport, sugar metabolism, mitogenome structure and dependence of viability on the mitogenome (the petite-negative phenotype). Transcriptions of these genomes produce only a few polycistronic transcripts, which then undergo processing as per the tRNA punctuation model. In general, the machinery for mitochondrial gene expression is structurally and functionally conserved between fission yeast and humans. Furthermore, molecular research on S. pombe is supported by a considerable number of experimental techniques and database resources. Owing to these advantages, fission yeast has significantly contributed to biomedical and fundamental research. Here, we review the current state of knowledge regarding S. pombe mitochondrial gene expression, and emphasise the pertinence of fission yeast as both a model and tool, especially for studies on mitochondrial translation.
    Keywords:  OXPHOS complexes; PPR proteins; Schizosaccharomyces pombe; gene expression; gene fusions; mitochondrial biogenesis; mitoribosome
    DOI:  https://doi.org/10.1002/iub.2801
  25. Clin Chim Acta. 2023 Dec 13. pii: S0009-8981(23)00513-2. [Epub ahead of print] 117711
    Impact of platelet activation on the release of cell-free mitochondria and circulating mitochondrial DNA.
    Benoit Roch, Ekaterina Pisareva, Alexia Mirandola, Cynthia Sanchez, Brice Pastor, Rita Tanos, Florence Frayssinoux, Mona Diab-Assaf, Philippe Anker, Zahra Al Amir Dache, Alain R Thierry.
       BACKGROUND: Research on circulating mitochondrial DNA (cir-mtDNA) based diagnostic is insufficient, as to its function, origin, structural features, and particularly its standardization of isolation. To date, plasma preparation performed in previous studies do not take into consideration the potential bias resulting from the release of mitochondria by activated platelets.
    METHODS: To tackle this, we compared the mtDNA amount determined by a standard plasma preparation method or a method optimally avoiding platelet activation. MtDNA extracted from the plasma of seven healthy individuals was quantified by Q-PCR in the course of the process of both methods submitted to filtration, freezing or differential centrifugation.
    RESULTS: 98.7 to 99.4% of plasma mtDNA corresponded to extracellular mitochondria, either free or into large extracellular vesicles. Without platelet activation, the proportion of both types of entities remained preponderant (76-80%), but the amount of detected mtDNA decreased 67-fold.
    CONCLUSION: We show the high capacity of platelets to release free mitochondria in "in vitro" conditions. This represents a potent confounding factor when extracting mtDNA for cir-mtDNA investigation. Platelet activation during pre-analytical conditions should therefore be avoided when studying cir-mtDNA. Our findings lead to a profound revision of the assumptions previously made by most works in this field. Overall, our data suggest the need to characterize or isolate mtDNA associated various structural forms, as well as to standardize plasma preparation, to better circumscribe cir-mtDNA's diagnostic capacity.
    Keywords:  circulating DNA; circulating mitochondria; mitochondria; plasma; platelet; quantitative PCR
    DOI:  https://doi.org/10.1016/j.cca.2023.117711
  26. Cancer Commun (Lond). 2023 Dec 22.
    Glucose-mediated mitochondrial reprogramming by cholesterol export at TM4SF5-enriched mitochondria-lysosome contact sites.
    Ji Eon Kim, So-Young Park, Chulhwan Kwak, Yoonji Lee, Dae-Geun Song, Jae Woo Jung, Haesong Lee, Eun-Ae Shin, Yangie Pinanga, Kyung-Hee Pyo, Eun Hae Lee, Wonsik Kim, Soyeon Kim, Chang-Duck Jun, Jeanho Yun, Sun Choi, Hyun-Woo Rhee, Kwang-Hyeon Liu, Jung Weon Lee.
       BACKGROUND: Transmembrane 4 L six family member 5 (TM4SF5) translocates subcellularly and functions metabolically, although it is unclear how intracellular TM4SF5 translocation is linked to metabolic contexts. It is thus of interests to understand how the traffic dynamics of TM4SF5 to subcellular endosomal membranes are correlated to regulatory roles of metabolisms.
    METHODS: Here, we explored the metabolic significance of TM4SF5 localization at mitochondria-lysosome contact sites (MLCSs), using in vitro cells and in vivo animal systems, via approaches by immunofluorescence, proximity labelling based proteomics analysis, organelle reconstitution etc. RESULTS: Upon extracellular glucose repletion following depletion, TM4SF5 became enriched at MLCSs via an interaction between mitochondrial FK506-binding protein 8 (FKBP8) and lysosomal TM4SF5. Proximity labeling showed molecular clustering of phospho-dynamic-related protein I (DRP1) and certain mitophagy receptors at TM4SF5-enriched MLCSs, leading to mitochondrial fission and autophagy. TM4SF5 bound NPC intracellular cholesterol transporter 1 (NPC1) and free cholesterol, and mediated export of lysosomal cholesterol to mitochondria, leading to impaired oxidative phosphorylation but intact tricarboxylic acid (TCA) cycle and β-oxidation. In mouse models, hepatocyte Tm4sf5 promoted mitophagy and cholesterol transport to mitochondria, both with positive relations to liver malignancy.
    CONCLUSIONS: Our findings suggested that TM4SF5-enriched MLCSs regulate glucose catabolism by facilitating cholesterol export for mitochondrial reprogramming, presumably while hepatocellular carcinogenesis, recapitulating aspects for hepatocellular carcinoma metabolism with mitochondrial reprogramming to support biomolecule synthesis in addition to glycolytic energetics.
    Keywords:  cholesterol; fluorescent imaging; glucose catabolism; hepatocellular carcinogenesis; membrane contact sites; mitochondria function; mitophagy; oxidative phosphorylation; protein-protein interaction; tetraspanin
    DOI:  https://doi.org/10.1002/cac2.12510
  27. Metabolites. 2023 Dec 12. pii: 1196. [Epub ahead of print]13(12):
    Metabolic Alterations in NADSYN1-Deficient Cells.
    Nils W F Meijer, Johan Gerrits, Susan Zwakenberg, Fried J T Zwartkruis, Nanda M Verhoeven-Duif, Judith J M Jans.
      NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid. We demonstrate that when precursors of the NAD+ salvage pathway in the form of nicotinamide become limiting, NADSYN1 deficiency results in a decline in intracellular NAD+ levels even in the presence of other potential NAD+ sources such as tryptophan and nicotinic acid. As a consequence, alterations in 122 and 69 metabolites are observed in NADSYN1-deficient A549 and HEK293T cells compared to the wild-type cell line (FC > 2 and p < 0.05). We thus show that NADSYN1 deficiency results in a metabolic phenotype characterized by alterations in glycolysis, the TCA cycle, the pentose phosphate pathway, and the polyol pathway.
    Keywords:  NAD+ deficiency; NAD+ salvage pathway; NADSYN1; Preiss–Handler pathway; de novo NAD+ synthesis pathway; glycolysis; metabolomics; pentose phosphate pathway; polyol pathway
    DOI:  https://doi.org/10.3390/metabo13121196
  28. Discov Nano. 2023 Dec 21. 18(1): 161
    One-pot, degradable, silica nanocarriers with encapsulated oligonucleotides for mitochondrial specific targeting.
    Chloe Trayford, Alissa Wilhalm, Pamela Habibovic, Hubert Smeets, Florence van Tienen, Sabine van Rijt.
      Mutations in nuclear and mitochondrial genes are responsible for severe chronic disorders such as mitochondrial myopathies. Gene therapy using antisense oligonucleotides is a promising strategy to treat mitochondrial DNA (mtDNA) diseases by blocking the replication of the mutated mtDNA. However, transport vehicles are needed for intracellular, mitochondria-specific transport of oligonucleotides. Nanoparticle (NP) based vectors such as large pore mesoporous silica nanoparticles (LP) often rely on surface complexation of oligonucleotides exposing them to nucleases and limiting mitochondria targeting and controlled release ability. In this work, stable, fluorescent, hollow silica nanoparticles (HSN) that encapsulate and protect oligonucleotides in the hollow core were synthesized by a facile one-pot procedure. Both rhodamine B isothiocyanate and bis[3-(triethoxysilyl)propyl]tetrasulfide were incorporated in the HSN matrix by co-condensation to enable cell tracing, intracellular-specific degradation and controlled oligonucleotide release. We also synthesized LP as a benchmark to compare the oligonucleotide loading and release efficacy of our HSN. Mitochondria targeting was enabled by NP functionalization with cationic, lipophilic Triphenylphosphine (TPP) and, for the first time a fusogenic liposome based carrier, previously reported under the name MITO-Porter. HSN exhibited high oligonucleotide incorporation ratios and release dependent on intracellular degradation. Further, MITO-Porter capping of our NP enabled delayed, glutathione (GSH) responsive oligonucleotide release and mitochondria targeting at the same efficiency as TPP functionalized NP. Overall, our NP are promising vectors for anti-gene therapy of mtDNA disease as well as many other monogenic disorders worldwide.
    Keywords:  Anti-gene therapy; Hollow silica nanoparticles; Intracellular degradation; Large pore silica nanoparticles; Mitochondria targeting
    DOI:  https://doi.org/10.1186/s11671-023-03926-1
  29. Kidney Int Rep. 2023 Dec;8(12): 2830-2832
    Quantitative Analysis of Mitochondrial DNA Heteroplasmy in Urinary Podocytes of Myoclonus Epilepsy With Ragged-Red Fibers Syndrome.
    Miwa Goto, Emi Sawanobori, Takeshi Inukai, Shuji Hirata, Tadashi Mabuchi.
      
    Keywords:  fragment analysis; heteroplasmy; mitochondrial diseases; proteinuria; standard curve; urinary podocytes
    DOI:  https://doi.org/10.1016/j.ekir.2023.09.021
  30. J Biol Chem. 2023 Nov 22. pii: S0021-9258(23)02498-5. [Epub ahead of print]300(1): 105470
    Complex II ambiguities-FADH2 in the electron transfer system.
    Erich Gnaiger.
      The prevailing notion that reduced cofactors NADH and FADH2 transfer electrons from the tricarboxylic acid cycle to the mitochondrial electron transfer system creates ambiguities regarding respiratory Complex II (CII). CII is the only membrane-bound enzyme in the tricarboxylic acid cycle and is part of the electron transfer system of the mitochondrial inner membrane feeding electrons into the coenzyme Q-junction. The succinate dehydrogenase subunit SDHA of CII oxidizes succinate and reduces the covalently bound prosthetic group FAD to FADH2 in the canonical forward tricarboxylic acid cycle. However, several graphical representations of the electron transfer system depict FADH2 in the mitochondrial matrix as a substrate to be oxidized by CII. This leads to the false conclusion that FADH2 from the β-oxidation cycle in fatty acid oxidation feeds electrons into CII. In reality, dehydrogenases of fatty acid oxidation channel electrons to the Q-junction but not through CII. The ambiguities surrounding Complex II in the literature and educational resources call for quality control, to secure scientific standards in current communications of bioenergetics, and ultimately support adequate clinical applications. This review aims to raise awareness of the inherent ambiguity crisis, complementing efforts to address the well-acknowledged issues of credibility and reproducibility.
    Keywords:  Complex II; coenzyme Q; electron transfer system; fatty acid oxidation; flavin adenine dinucleotide; succinate dehydrogenase; tricarboxylic acid cycle
    DOI:  https://doi.org/10.1016/j.jbc.2023.105470
  31. Free Radic Biol Med. 2023 Dec 13. pii: S0891-5849(23)01157-7. [Epub ahead of print]
    Mitochondrial transplantation attenuates oligomeric amyloid-beta-induced mitochondrial dysfunction and tight junction protein destruction in retinal pigment epithelium.
    Sung-Eun Noh, Seok Jae Lee, Chang Sik Cho, Dong Hyun Jo, Kyu Sang Park, Jeong Hun Kim.
      Transplantation of mitochondria derived from mesenchymal stem cells (MSCs) has emerged as a new treatment method to improve mitochondrial dysfunction and alleviate cell impairment. Interest in using extrinsic mitochondrial transplantation as a therapeutic approach has been increasing because it has been confirmed to be effective in treating various diseases related to mitochondrial dysfunction, including ischemia, cardiovascular disease, and toxic damage. To support this application, we conducted an experiment to deliver external mitochondria to retinal pigment epithelial cells treated with oligomeric amyloid-beta (oAβ). Externally delivered amyloid-beta internalizes into cells and interacts with mitochondria, resulting in mitochondrial dysfunction and intracellular damage, including increased reactive oxygen species and destruction of tight junction proteins. Externally delivered mitochondria were confirmed to alleviate mitochondrial dysfunction and tight junction protein disruption as well as improve internalized oAβ clearance. These results were also confirmed in a mouse model in vivo. Overall, these findings indicate that the transfer of external mitochondria isolated from MSCs has potential as a new treatment method for age-related macular degeneration, which involves oAβ-induced changes to the retinal pigment epithelium.
    Keywords:  Age-related macular degeneration; Mesenchymal stem cells; Mitochondrial transplantation; Oligomeric amyloid-beta; Reactive oxygen species; Tight junction proteins
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.12.012
  32. Neuroradiology. 2023 Dec 19.
    Magnetic resonance spectroscopy in MELAS syndrome: correlation with CSF and plasma metabolite levels and change after glutamine supplementation.
    María Paz Guerrero-Molina, Ángela Bernabeu-Sanz, Ana Ramos-González, Montserrat Morales-Conejo, Aitor Delmiro, Cristina Domínguez-González, Joaquín Arenas, Miguel A Martín, Jesús González de la Aleja.
       PURPOSE: MELAS syndrome is a genetic disorder caused by mitochondrial DNA mutations. We previously described that MELAS patients had increased CSF glutamate and decreased CSF glutamine levels and that oral glutamine supplementation restores these values. Proton magnetic resonance spectroscopy (1H-MRS) allows the in vivo evaluation of brain metabolism. We aimed to compare 1H-MRS of MELAS patients with controls, the 1H-MRS after glutamine supplementation in the MELAS group, and investigate the association between 1H-MRS and CSF lactate, glutamate, and glutamine levels.
    METHODS: We conducted an observational case-control study and an open-label, single-cohort study with single-voxel MRS (TE 144/35 ms). We assessed the brain metabolism changes in the prefrontal (PFC) and parieto-occipital) cortex (POC) after oral glutamine supplementation in MELAS patients. MR spectra were analyzed with jMRUI software.
    RESULTS: Nine patients with MELAS syndrome (35.8 ± 3.2 years) and nine sex- and age-matched controls were recruited. Lactate/creatine levels were increased in MELAS patients in both PFC and POC (0.40 ± 0.05 vs. 0, p < 0.001; 0.32 ± 0.03 vs. 0, p < 0.001, respectively). No differences were observed between groups in glutamate and glutamine (Glx/creatine), either in PFC (p = 0.930) or POC (p = 0.310). No differences were observed after glutamine supplementation. A positive correlation was found between CSF lactate and lactate/creatine only in POC (0.85, p = 0.003).
    CONCLUSION: No significant metabolite changes were observed in the brains of MELAS patients after glutamine supplementation. While we found a positive correlation between lactate levels in CSF and 1H-MRS in MELAS patients, we could not monitor treatment response over short periods with this tool.
    TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04948138; initial release 24/06/2021; first patient enrolled on 1/07/2021. https://clinicaltrials.gov/ct2/show/NCT04948138.
    Keywords:  Glutamate; Glutamine supplementation; MELAS syndrome; MRI spectroscopy; Mitochondrial disease
    DOI:  https://doi.org/10.1007/s00234-023-03263-1
  33. J Med Chem. 2023 Dec 19.
    Cyclohexylalanine-Containing α-Helical Amphipathic Peptide Targets Cardiolipin, Rescuing Mitochondrial Dysfunction in Kidney Injury.
    Gwangsu Shin, Soonsil Hyun, Dongwoo Kim, Yoonhwa Choi, Kyu Hong Kim, Dongmin Kim, Soie Kwon, Yon Su Kim, Seung Hee Yang, Jaehoon Yu.
      Mitochondrial dysfunction is linked to degenerative diseases, resulting from cardiolipin (CL)-induced disruption of cristae structure in the inner mitochondrial membrane (IMM); therefore, preserving cristae and preventing CL remodeling offer effective strategies to maintain mitochondrial function. To identify reactive oxygen species (ROS)-blocking agents against mitochondrial dysfunction, a library of cyclohexylamine-containing cell-penetrating α-helical amphipathic "bundle" peptides were screened. Among these, CMP3013 is selectively bound to abnormal mitochondria, preserving the cristae structure impaired by mitochondria-damaging agents. With a stronger affinity for CL compared with other IMM lipid components, CMP3013 exhibited high selectivity. Consequently, it protected cristae, reduced ROS production, and enhanced adenosine triphosphate (ATP) generation. In mouse models of acute kidney injury, a 1 mg/kg dose of CMP3013 demonstrated remarkable efficacy, highlighting its potential as a therapeutic agent for mitochondrial dysfunction-related disorders. Overall, CMP3013 represents a promising agent for mitigating mitochondrial dysfunction and associated diseases.
    DOI:  https://doi.org/10.1021/acs.jmedchem.3c01578
  34. Genes Cells. 2023 Dec 22.
    Generation of inducible mitophagy mice.
    Toshiyuki Nishiji, Atsushi Hoshino, Yuki Uchio, Satoaki Matoba.
      Mitophagy is programmed selective autophagy of mitochondria and is important for mitochondrial quality control and cellular homeostasis. Mitochondrial dysfunction and impaired mitophagy are closely associated with various diseases, including heart failure and diabetes. To better understand the pathophysiological role of mitophagy, we generated doxycycline-inducible mitophagy mice using a synthetic mitophagy adaptor protein consisting of an outer mitochondrial membrane targeting sequence and an engineered LIR. To evaluate the activation of mitophagy upon doxycycline treatment, we also generated mitophagy reporter mito-QC mice in which mitochondria tandemly express mCherry and GFP, and only GFP signals are lost in acidic lysosomes subjected to mitophagy. With the ROSA26 promoter-driven rtTA, mitophagy was observed at least in heart, liver, and skeletal muscle. We investigated the relationship between mitophagy activation and pressure overload heart failure or high fat diet-induced obesity. Unexpectedly, we were unable to confirm the protective effect of mitophagy in these two pathological models. Further titration of the level of mitophagy induction is required to demonstrate the potency of the protective effects of mitophagy in disease models.
    Keywords:  bioengineering; heart failure; mitophagy
    DOI:  https://doi.org/10.1111/gtc.13091
  35. Am J Hum Genet. 2023 Dec 12. pii: S0002-9297(23)00404-4. [Epub ahead of print]
    ClinGen guidance for use of the PP1/BS4 co-segregation and PP4 phenotype specificity criteria for sequence variant pathogenicity classification.
    ClinGen Sequence Variant Interpretation Working Group
      The 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology variant classification publication established a standard employed internationally to guide laboratories in variant assessment. Those recommendations included both pathogenic (PP1) and benign (BS4) criteria for evaluating the inheritance patterns of variants, but details of how to apply those criteria at appropriate evidence levels were sparse. Several publications have since attempted to provide additional guidance, but anecdotally, this issue is still challenging. Additionally, it is not clear that those prior efforts fully distinguished disease-gene identification considerations from variant pathogenicity considerations nor did they address autosomal-recessive and X-linked inheritance. Here, we have taken a mixed inductive and deductive approach to this problem using real diseases as examples. We have developed a practical heuristic for genetic co-segregation evidence and have also determined that the specific phenotype criterion (PP4) is inseparably coupled to the co-segregation criterion. We have also determined that negative evidence at one locus constitutes positive evidence for other loci for disorders with locus heterogeneity. Finally, we provide a points-based system for evaluating phenotype and co-segregation as evidence types to support or refute a locus and show how that can be integrated into the Bayesian framework now used for variant classification and consistent with the 2015 guidelines.
    DOI:  https://doi.org/10.1016/j.ajhg.2023.11.009
  36. Cells. 2023 Dec 07. pii: 2781. [Epub ahead of print]12(24):
    Disruption of Mitophagy Flux through the PARL-PINK1 Pathway by CHCHD10 Mutations or CHCHD10 Depletion.
    Tian Liu, Liam Wetzel, Zexi Zhu, Pavan Kumaraguru, Viraj Gorthi, Yan Yan, Mohammed Zaheen Bukhari, Aizara Ermekbaeva, Hanna Jeon, Teresa R Kee, Jung-A Alexa Woo, David E Kang.
      Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) is a nuclear-encoded mitochondrial protein which is primarily mutated in the spectrum of familial and sporadic amyotrophic lateral sclerosis (ALS)-frontotemporal dementia (FTD). Endogenous CHCHD10 levels decline in the brains of ALS-FTD patients, and the CHCHD10S59L mutation in Drosophila induces dominant toxicity together with PTEN-induced kinase 1 (PINK1), a protein critical for the induction of mitophagy. However, whether and how CHCHD10 variants regulate mitophagy flux in the mammalian brain is unknown. Here, we demonstrate through in vivo and in vitro models, as well as human FTD brain tissue, that ALS/FTD-linked CHCHD10 mutations (R15L and S59L) impair mitophagy flux and mitochondrial Parkin recruitment, whereas wild-type CHCHD10 (CHCHD10WT) normally enhances these measures. Specifically, we show that CHCHD10R15L and CHCHD10S59L mutations reduce PINK1 levels by increasing PARL activity, whereas CHCHD10WT produces the opposite results through its stronger interaction with PARL, suppressing its activity. Importantly, we also demonstrate that FTD brains with TAR DNA-binding protein-43 (TDP-43) pathology demonstrate disruption of the PARL-PINK1 pathway and that experimentally impairing mitophagy promotes TDP-43 aggregation. Thus, we provide herein new insights into the regulation of mitophagy and TDP-43 aggregation in the mammalian brain through the CHCHD10-PARL-PINK1 pathway.
    Keywords:  CHCHD10; PARL; PINK1; TDP-43; mitophagy
    DOI:  https://doi.org/10.3390/cells12242781
  37. Circulation. 2023 Dec 21.
    Ago2 Protects Against Diabetic Cardiomyopathy by Activating Mitochondrial Gene Translation.
    Jiabing Zhan, Kunying Jin, Rong Xie, Jiahui Fan, Yuyan Tang, Chen Chen, Huaping Li, Dao Wen Wang.
       BACKGROUND: Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular Ago2 (Argonaute2), a core member of miRNA machinery, remain elusive.
    METHODS: We elucidated the function and mechanism of subcellular localized Ago2 on mouse models for diabetes and diabetic cardiomyopathy. Recombinant adeno-associated virus type 9 was used to deliver Ago2 to mice through the tail vein. Cardiac structure and functions were assessed by echocardiography and catheter manometer system.
    RESULTS: Ago2 was decreased in mitochondria of diabetic cardiomyocytes. Overexpression of mitochondrial Ago2 attenuated diabetes-induced cardiac dysfunction. Ago2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain subunits and decrease reactive oxygen species. Malonylation, a posttranslational modification of Ago2, reduced the importing of Ago2 into mitochondria in diabetic cardiomyopathy. Ago2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function.
    CONCLUSIONS: Our findings reveal that the SIRT3-Ago2-CYTB axis links glucotoxicity to cardiac electron transport chain imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.
    Keywords:  diabetic cardiomyopathies; mitochondria; therapeutics
    DOI:  https://doi.org/10.1161/CIRCULATIONAHA.123.065546
  38. Behav Brain Res. 2023 Dec 14. pii: S0166-4328(23)00529-6. [Epub ahead of print] 114811
    Mitochondrial homeostasis regulation: A promising therapeutic target for Parkinson's disease.
    Meng-Fan Yao, Tao Dang, Hua-Jun Wang, Xiao-Zhong Zhu, Chen Qiao.
      Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies (LBs) or Lewy neurites (LNs) which consist of α-synuclein (α-syn) and a complex mix of other biomolecules. Mitochondrial dysfunction is widely believed to play an essential role in the pathogenesis of PD and other related neurodegenerative diseases. But mitochondrial dysfunction is subject to complex genetic regulation. There is increasing evidence that PD-related genes directly or indirectly affect mitochondrial integrity. Therefore, targeted regulation of mitochondrial function has great clinical application prospects in the treatment of PD. However, lots of PD drugs targeting mitochondria have been developed but their clinical therapeutic effects are not ideal. This review aims to reveal the role of mitochondrial dysfunction in the pathogenesis of neurodegenerative diseases based on the mitochondrial structure and function, which may highlight potential interventions and therapeutic targets for the development of PD drugs to recover mitochondrial dysfunction in neurodegenerative diseases.
    Keywords:  DNA damage; Mitochondrial dynamics; Mitochondrial homeostasis; Mitochondrial quality control; Parkinson’s disease; Pharmaceutical research and development
    DOI:  https://doi.org/10.1016/j.bbr.2023.114811
  39. Nucleic Acids Res. 2023 Dec 20. pii: gkad1204. [Epub ahead of print]
    Light-strand bias and enriched zones of embedded ribonucleotides are associated with DNA replication and transcription in the human-mitochondrial genome.
    Penghao Xu, Taehwan Yang, Deepali L Kundnani, Mo Sun, Stefania Marsili, Alli L Gombolay, Youngkyu Jeon, Gary Newnam, Sathya Balachander, Veronica Bazzani, Umberto Baccarani, Vivian S Park, Sijia Tao, Adriana Lori, Raymond F Schinazi, Baek Kim, Zachary F Pursell, Gianluca Tell, Carlo Vascotto, Francesca Storici.
      Abundant ribonucleoside-triphosphate (rNTP) incorporation into DNA by DNA polymerases in the form of ribonucleoside monophosphates (rNMPs) is a widespread phenomenon in nature, resulting in DNA-structural change and genome instability. The rNMP distribution, characteristics, hotspots and association with DNA metabolic processes in human mitochondrial DNA (hmtDNA) remain mostly unknown. Here, we utilize the ribose-seq technique to capture embedded rNMPs in hmtDNA of six different cell types. In most cell types, the rNMPs are preferentially embedded on the light strand of hmtDNA with a strong bias towards rCMPs; while in the liver-tissue cells, the rNMPs are predominately found on the heavy strand. We uncover common rNMP hotspots and conserved rNMP-enriched zones across the entire hmtDNA, including in the control region, which links the rNMP presence to the frequent hmtDNA replication-failure events. We show a strong correlation between coding-sequence size and rNMP-embedment frequency per nucleotide on the non-template, light strand in all cell types, supporting the presence of transient RNA-DNA hybrids preceding light-strand replication. Moreover, we detect rNMP-embedment patterns that are only partly conserved across the different cell types and are distinct from those found in yeast mtDNA. The study opens new research directions to understand the biology of hmtDNA and genomic rNMPs.
    DOI:  https://doi.org/10.1093/nar/gkad1204
  40. Comput Biol Med. 2023 Dec 13. pii: S0010-4825(23)01275-1. [Epub ahead of print]169 107810
    An integrative framework for clinical diagnosis and knowledge discovery from exome sequencing data.
    Mona Shojaei, Navid Mohammadvand, Tunca Doğan, Can Alkan, Rengül Çetin Atalay, Aybar C Acar.
      Non-silent single nucleotide genetic variants, like nonsense changes and insertion-deletion variants, that affect protein function and length substantially are prevalent and are frequently misclassified. The low sensitivity and specificity of existing variant effect predictors for nonsense and indel variations restrict their use in clinical applications. We propose the Pathogenic Mutation Prediction (PMPred) method to predict the pathogenicity of single nucleotide variations, which impair protein function by prematurely terminating a protein's elongation during its synthesis. The prediction starts by monitoring functional effects (Gene Ontology annotation changes) of the change in sequence, using an existing ensemble machine learning model (UniGOPred). This, in turn, reveals the mutations that significantly deviate functionally from the wild-type sequence. We have identified novel harmful mutations in patient data and present them as motivating case studies. We also show that our method has increased sensitivity and specificity compared to state-of-the-art, especially in single nucleotide variations that produce large functional changes in the final protein. As further validation, we have done a comparative docking study on such a variation that is misclassified by existing methods and, using the altered binding affinities, show how PMPred can correctly predict the pathogenicity when other tools miss it. PMPred is freely accessible as a web service at https://pmpred.kansil.org/, and the related code is available at https://github.com/kansil/PMPred.
    Keywords:  Gene ontology; Insertion-deletion variants; Mutation; Pearson correlation; Protein sequence; Variant pathogenicity prediction
    DOI:  https://doi.org/10.1016/j.compbiomed.2023.107810
  41. J Am Med Inform Assoc. 2023 Dec 22. pii: ocad244. [Epub ahead of print]
    Automatically pre-screening patients for the rare disease aromatic l-amino acid decarboxylase deficiency using knowledge engineering, natural language processing, and machine learning on a large EHR population.
    Aaron M Cohen, Jolie Kaner, Ryan Miller, Jeffrey W Kopesky, William Hersh.
       OBJECTIVES: Electronic health record (EHR) data may facilitate the identification of rare diseases in patients, such as aromatic l-amino acid decarboxylase deficiency (AADCd), an autosomal recessive disease caused by pathogenic variants in the dopa decarboxylase gene. Deficiency of the AADC enzyme results in combined severe reductions in monoamine neurotransmitters: dopamine, serotonin, epinephrine, and norepinephrine. This leads to widespread neurological complications affecting motor, behavioral, and autonomic function. The goal of this study was to use EHR data to identify previously undiagnosed patients who may have AADCd without available training cases for the disease.
    MATERIALS AND METHODS: A multiple symptom and related disease annotated dataset was created and used to train individual concept classifiers on annotated sentence data. A multistep algorithm was then used to combine concept predictions into a single patient rank value.
    RESULTS: Using an 8000-patient dataset that the algorithms had not seen before ranking, the top and bottom 200 ranked patients were manually reviewed for clinical indications of performing an AADCd diagnostic screening test. The top-ranked patients were 22.5% positively assessed for diagnostic screening, with 0% for the bottom-ranked patients. This result is statistically significant at P < .0001.
    CONCLUSION: This work validates the approach that large-scale rare-disease screening can be accomplished by combining predictions for relevant individual symptoms and related conditions which are much more common and for which training data is easier to create.
    Keywords:  EHR data secondary uses; aromatic l-amino acid decarboxylase deficiency; machine learning; natural language processing; rare diseases
    DOI:  https://doi.org/10.1093/jamia/ocad244
  42. Int J Mol Sci. 2023 Dec 15. pii: 17503. [Epub ahead of print]24(24):
    Translation Fidelity and Respiration Deficits in CLPP-Deficient Tissues: Mechanistic Insights from Mitochondrial Complexome Profiling.
    Jana Key, Suzana Gispert, Gabriele Koepf, Julia Steinhoff-Wagner, Marina Reichlmeir, Georg Auburger.
      The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5'-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.
    Keywords:  azoospermia; mtLSU quality control; primary ovarian insufficiency; rRNA chaperone ERAL1; sensorineural hearing loss
    DOI:  https://doi.org/10.3390/ijms242417503
  43. Mol Genet Metab Rep. 2024 Mar;38 101025
    Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL): Report of a new family and a novel DARS2 mutation.
    Wei-Lin Huang, Maija R Steenari, Rebekah Barrick, Mariella T Simon, Richard Chang, Shaya S Eftekharian, Alexander Stover, Philip H Schwartz, Alexandra Latini, Jose E Abdenur.
       Background: LBSL is a mitochondrial disorder caused by mutations in the mitochondrial aspartyl-tRNA synthetase gene DARS2, resulting in a distinctive pattern on brain magnetic resonance imaging (MRI) and spectroscopy. Clinical presentation varies from severe infantile to chronic, slowly progressive neuronal deterioration in adolescents or adults. Most individuals with LBSL are compound heterozygous for one splicing defect in an intron 2 mutational hotspot and a second defect that could be a missense, non-sense, or splice site mutation or deletion resulting in decreased expression of the full-length protein.
    Aim: To present a new family with two affected members with LBSL and report a novel DARS2 mutation.
    Results: An 8-year-old boy (Patient 1) was referred due to headaches and abnormal MRI, suggestive of LBSL. Genetic testing revealed a previously reported c.492 + 2 T > C mutation in the DARS2 gene. Sanger sequencing uncovered a novel variant c.228-17C > G in the intron 2 hotspot. Family studies found the same genetic changes in an asymptomatic 4-year-old younger brother (Patient 2), who was found on follow-up to have an abnormal MRI. mRNA extracted from patients' fibroblasts showed that the c.228-17C > G mutation caused skipping of exon 3 resulting in lower DARS2 mRNA level. Complete absence of DARS2 protein was also found in both patients.
    Summary: We present a new family with two children affected with LBSL and describe a novel mutation in the DARS2 intron 2 hotspot. Despite findings of extensive white matter disease in the brain and spine, the proband in this family presented only with headaches, while the younger sibling, who also had extensive white matter changes, was asymptomatic. Our in-vitro results confirmed skipping of exon 3 in patients and family members carrying the intron 2 variant, which is consistent with previous reported mutations in intron 2 hotspots. DARS2 mRNA and protein levels were also reduced in both patients, further supporting the pathogenicity of the novel variant.
    Keywords:  Aminoacyl tRNA synthetase deficiency; Aspartyl tRNA synthetase deficiency; Leukodystrophy; Mitochondrial disorders; Splicing mutations
    DOI:  https://doi.org/10.1016/j.ymgmr.2023.101025
  44. Nat Cell Biol. 2023 Dec 21.
    Dissecting organelle interdependence.
    Antentor Hinton, Andrea G Marshall.
      
    DOI:  https://doi.org/10.1038/s41556-023-01285-8
  45. Genome Biol. 2023 Dec 21. 24(1): 294
    Benchmarking splice variant prediction algorithms using massively parallel splicing assays.
    Cathy Smith, Jacob O Kitzman.
       BACKGROUND: Variants that disrupt mRNA splicing account for a sizable fraction of the pathogenic burden in many genetic disorders, but identifying splice-disruptive variants (SDVs) beyond the essential splice site dinucleotides remains difficult. Computational predictors are often discordant, compounding the challenge of variant interpretation. Because they are primarily validated using clinical variant sets heavily biased to known canonical splice site mutations, it remains unclear how well their performance generalizes.
    RESULTS: We benchmark eight widely used splicing effect prediction algorithms, leveraging massively parallel splicing assays (MPSAs) as a source of experimentally determined ground-truth. MPSAs simultaneously assay many variants to nominate candidate SDVs. We compare experimentally measured splicing outcomes with bioinformatic predictions for 3,616 variants in five genes. Algorithms' concordance with MPSA measurements, and with each other, is lower for exonic than intronic variants, underscoring the difficulty of identifying missense or synonymous SDVs. Deep learning-based predictors trained on gene model annotations achieve the best overall performance at distinguishing disruptive and neutral variants, and controlling for overall call rate genome-wide, SpliceAI and Pangolin have superior sensitivity. Finally, our results highlight two practical considerations when scoring variants genome-wide: finding an optimal score cutoff, and the substantial variability introduced by differences in gene model annotation, and we suggest strategies for optimal splice effect prediction in the face of these issues.
    CONCLUSION: SpliceAI and Pangolin show the best overall performance among predictors tested, however, improvements in splice effect prediction are still needed especially within exons.
    DOI:  https://doi.org/10.1186/s13059-023-03144-z
  46. Brain Dev. 2023 Dec 21. pii: S0387-7604(23)00195-X. [Epub ahead of print]
    A comprehensive study of mutation and phenotypic heterogeneity of childhood mitochondrial leukodystrophies.
    Sareh Hosseinpour, Ehsan Razmara, Morteza Heidari, Zahra Rezaei, Mahmoud Reza Ashrafi, Ali Zare Dehnavi, Reyhaneh Kameli, Ali Hosseini Bereshneh, Hassan Vahidnezhad, Reza Azizimalamiri, Zahra Zamani, Neda Pak, Maryam Rasulinezhad, Bahram Mohammadi, Homa Ghabeli, Mohammad Ghafouri, Mahmoud Mohammadi, Gholam Reza Zamani, Reza Shervin Badv, Sasan Saket, Bahareh Rabbani, Nejat Mahdieh, Ali Ahani, Masoud Garshasbi, Ali Reza Tavasoli.
       OBJECTIVE: Mitochondrial leukodystrophies (MLs) are mainly caused by impairments of the mitochondrial respiratory chains. This study reports the mutation and phenotypic spectrum of a cohort of 41 pediatric patients from 39 distinct families with MLs among 320 patients with a molecular diagnosis of leukodystrophies.
    METHODS: This study summarizes the clinical, imaging, and molecular data of these patients for five years.
    RESULTS: The three most common symptoms were neurologic regression (58.5%), pyramidal signs (58.5%), and extrapyramidal signs (43.9%). Because nuclear DNA mutations are responsible for a high percentage of pediatric MLs, whole exome sequencing was performed on all patients. In total, 39 homozygous variants were detected. Additionally, two previously reported mtDNA variants were identified with different levels of heteroplasmy in two patients. Among 41 mutant alleles, 33 (80.4%) were missense, 4 (9.8%) were frameshift (including 3 deletions and one duplication), and 4 (9.8%) were splicing mutations. Oxidative phosphorylation in 27 cases (65.8%) and mtDNA maintenance pathways in 8 patients (19.5%) were the most commonly affected mitochondrial pathways. In total, 5 novel variants in PDSS1, NDUFB9, FXBL4, SURF1, and NDUSF1 were also detected. In silico analyses showed how each novel variant may contribute to ML pathogenesis.
    CONCLUSIONS: The findings of this study suggest whole-exome sequencing as a strong diagnostic genetic tool to identify the causative variants in pediatric MLs. In comparison between oxidative phosphorylation (OXPHOS) and mtDNA maintenance groups, brain stem and periaqueductal gray matter (PAGM) involvement were more commonly seen in OXPHOS group (P value of 0.002 and 0.009, respectively), and thinning of corpus callosum was observed more frequently in mtDNA maintenance group (P value of 0.042).
    Keywords:  Children; Mitochondrial Leukodystrophy; Mutation; OXPHOS; Phenotype
    DOI:  https://doi.org/10.1016/j.braindev.2023.12.003
  47. Heliyon. 2023 Nov;9(11): e22459
    An overview of ATP synthase, inhibitors, and their toxicity.
    Arwa R Althaher, Mohammad Alwahsh.
      Mitochondrial complex V (ATP synthase) is a remarkable molecular motor crucial in generating ATP and sustaining mitochondrial function. Its importance in cellular metabolism cannot be overstated, as malfunction of ATP synthase has been linked to various pathological conditions. Both natural and synthetic ATP synthase inhibitors have been extensively studied, revealing their inhibitory sites and modes of action. These findings have opened exciting avenues for developing new therapeutics and discovering new pesticides and herbicides to safeguard global food supplies. However, it is essential to remember that these compounds can also adversely affect human and animal health, impacting vital organs such as the nervous system, heart, and kidneys. This review aims to provide a comprehensive overview of mitochondrial ATP synthase, its structural and functional features, and the most common inhibitors and their potential toxicities.
    Keywords:  ATP synthase; ATP synthase inhibitors; Electron transport chain; Proton motive force; Rotational catalysis
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e22459
  48. Nat Genet. 2023 Dec 18.
    Gene therapy for Parkinson's disease models.
    Tiago Faial.
      
    DOI:  https://doi.org/10.1038/s41588-023-01632-3
  49. Stem Cell Reports. 2023 Nov 27. pii: S2213-6711(23)00453-8. [Epub ahead of print]
    Human retinal organoids with an OPA1 mutation are defective in retinal ganglion cell differentiation and function.
    Qiannan Lei, Kangjian Xiang, Lin Cheng, Mengqing Xiang.
      Autosomal dominant optic atrophy (ADOA), mostly caused by heterozygous OPA1 mutations and characterized by retinal ganglion cell (RGC) loss and optic nerve degeneration, is one of the most common types of inherited optic neuropathies. Previous work using a two-dimensional (2D) differentiation model of induced pluripotent stem cells (iPSCs) has investigated ADOA pathogenesis but failed to agree on the effect of OPA1 mutations on RGC differentiation. Here, we use 3D retinal organoids capable of mimicking in vivo retinal development to resolve the issue. We generated isogenic iPSCs carrying the hotspot OPA1 c.2708_2711delTTAG mutation and found that the mutant variant caused defective initial and terminal differentiation and abnormal electrophysiological properties of organoid-derived RGCs. Moreover, this variant inhibits progenitor proliferation and results in mitochondrial dysfunction. These data demonstrate that retinal organoids coupled with gene editing serve as a powerful tool to definitively identify disease-related phenotypes and provide valuable resources to further investigate ADOA pathogenesis and screen for ADOA therapeutics.
    Keywords:  autosomal dominant optic atrophy; mitochondria; optic atrophy 1; retinal ganglion cell; retinal organoid
    DOI:  https://doi.org/10.1016/j.stemcr.2023.11.004
  50. Pract Neurol. 2023 Dec 22. pii: pn-2023-003917. [Epub ahead of print]
    Neurological gait assessment.
    Sofia Mermelstein, Pedro Barbosa, Diego Kaski.
      Gait disorders are a common feature of neurological disease. The gait examination is an essential part of the neurological clinical assessment, providing valuable clues to a myriad of causes. Understanding how to examine gait is not only essential for neurological diagnosis but also for treatment and prognosis. Here, we review aspects of the clinical history and examination of neurological gait to help guide gait disorder assessment. We focus particularly on how to differentiate between common gait abnormalities and highlight the characteristic features of the more prevalent neurological gait patterns such as ataxia, waddling, steppage, spastic gait, Parkinson's disease and functional gait disorders. We also offer diagnostic clues for some unusual gait presentations, such as dystonic, stiff-person and choreiform gait, along with red flags that help differentiate atypical parkinsonism from Parkinson's disease.
    Keywords:  APRAXIA; CEREBELLAR ATAXIA; CLINICAL NEUROLOGY; GAIT; PARKINSON-S DISEASE
    DOI:  https://doi.org/10.1136/pn-2023-003917
  51. Sci Rep. 2023 Dec 15. 13(1): 22326
    Structural insights into N-terminal methionine cleavage by the human mitochondrial methionine aminopeptidase, MetAP1D.
    Yeon Lee, Hayoung Kim, Eunji Lee, Hyunggu Hahn, Yoonyoung Heo, Dong Man Jang, Kihyuck Kwak, Hyo Jung Kim, Hyoun Sook Kim.
      Isozymes are enzymes that catalyze identical biological reactions, yet exhibit slight variations in structures and catalytic efficiency, which enables the precise adjustment of metabolism to fulfill the specific requirements of a particular tissue or stage of development. Methionine aminopeptidase (MetAP) isozymes function a critical role in cleaving N-terminal methionine from nascent proteins to generate functional proteins. In humans, two distinct MetAP types I and II have been identified, with type I further categorized into cytosolic (MetAP1) and mitochondrial (MetAP1D) variants. However, despite extensive structural studies on both bacterial and human cytosolic MetAPs, the structural information remains unavailable for human mitochondrial MetAP. This study was aimed to elucidate the high-resolution structures of human mitochondrial MetAP1D in its apo-, cobalt-, and methionine-bound states. Through a comprehensive analysis of the determined structures and a docking simulation model with mitochondrial substrate peptides, we present mechanistic insights into the cleavage process of the initiator methionine from mitochondrial proteins. Notably, despite the shared features at the active site between the cytosolic and mitochondrial MetAP type I isozymes, we identified distinct structural disparities within the active-site pocket primarily contributed by two specific loops that could play a role in accommodating specific substrates. These structural insights offer a basis for the further exploration of MetAP isozymes as critical players in cellular processes and potential therapeutic applications.
    DOI:  https://doi.org/10.1038/s41598-023-49332-6
  52. Anal Biochem. 2023 Dec 20. pii: S0003-2697(23)00410-4. [Epub ahead of print] 115445
    REAP+: A single preparation for rapid isolation of nuclei, cytoplasm, and mitochondria.
    Barbara Dos Santos, Monique Coelho Bion, Marie Goujon-Svrzic, Pamela Maher, Alcir Luiz Dafre.
      REAP+ is an enhanced version of the rapid, efficient, and practical (REAP) method designed for the isolation of nuclear fractions. This improved version, REAP+, enables fast and effective extraction of mitochondria, cytoplasm, and nuclei. The mechanical cell disruption process has been optimized to cerebral tissues, snap-frozen liver, and HT22 cells with remarkable fraction enrichment. REAP+ is well-suited for samples containing minimal protein quantities, such as mouse hippocampal slices. The method was validated by Western blot and marker enzyme activities, such as LDH and G6PDH for the cytoplasmic fraction and succinate dehydrogenase and cytochrome c oxidase for the mitochondrial fraction. One of the outstanding features of this method is its rapid execution, yielding fractions within 15 min, allowing for simultaneous preparation of multiple samples. In essence, REAP + emerges as a swift, efficient, and practical technique for the concurrent isolation of nuclei, cytoplasm, and mitochondria from various cell types and tissues. The method would be suitable to study the multicompartment translocation of proteins, such as metabolic enzymes and transcription factors migrating from cytosol to the mitochondria and nuclei. Moreover, its compatibility with small samples, such as hippocampal slices, and its potential applicability to human biopsies, highlights the potential application in medical research.
    Keywords:  Mitochondrial enrichment; Nuclear extract; Organelle-specific fractionation; REAP+; Subcellular fractionation
    DOI:  https://doi.org/10.1016/j.ab.2023.115445
  53. Mol Metab. 2023 Dec 15. pii: S2212-8778(23)00188-6. [Epub ahead of print] 101854
    Need for speed: Human fast-twitch mitochondria favor power over efficiency.
    Sebastian Edman, Mikael Flockhart, Filip J Larsen, William Apró.
       OBJECTIVE: Human skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation (OXPHOS). While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic.
    METHODS: Here, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within 6 h of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity.
    RESULTS: Maximal complex I CI+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for CI+II, which was primarily mediated through elevated CII respiration, but not CI or. Furthermore, the abundance of CII protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid-or complex I respiration.
    CONCLUSION: Mitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II.
    DOI:  https://doi.org/10.1016/j.molmet.2023.101854
  54. J Am Chem Soc. 2023 Dec 22.
    Real-Time Monitoring of Mitochondrial Damage Using Conjugated Oligoelectrolytes.
    Samuel J W Chan, Ji-Yu Zhu, Wilson Wee Mia Soh, Guillermo C Bazan.
      Conjugated oligoelectrolytes (COEs) comprise a class of fluorescent reporters with tunable optical properties and lipid bilayer affinity. These molecules have proven effective in a range of bioimaging applications; however, their use in characterizing specific subcellular structures remains restricted. Such capabilities would broaden COE applications to understand cellular dysfunction, cell communication, and the targets of different pharmaceutical agents. Here, we disclose a novel COE derivative, COE-CN, which enables the visualization of mitochondria, including morphological changes and lysosomal fusion upon treatment with depolarizing agents. COE-CN is characterized by the presence of imidazolium solubilizing groups and an optically active cyanovinyl-linked distyrylbenzene core with intramolecular charge-transfer characteristics. Our current understanding is that the relatively shorter molecular length of COE-CN leads to weaker binding within lipid bilayer membranes, which allows sampling of internal cellular structures and ultimately to different localization relative to elongated COEs. As a means of practical demonstration, COE-CN can be used to diagnose cells with damaged mitochondria via flow cytometry. Coupled with an elongated COE that does not translocate upon depolarization, changes in ratiometric fluorescence intensity can be used to monitor mitochondrial membrane potential disruption, demonstrating the potential for use in diagnostic assays.
    DOI:  https://doi.org/10.1021/jacs.3c10531
  55. Nat Protoc. 2023 Dec 22.
    Streamlining spatial omics data analysis with Pysodb.
    Senlin Lin, Fangyuan Zhao, Zihan Wu, Jianhua Yao, Yi Zhao, Zhiyuan Yuan.
      Advances in spatial omics technologies have improved the understanding of cellular organization in tissues, leading to the generation of complex and heterogeneous data and prompting the development of specialized tools for managing, loading and visualizing spatial omics data. The Spatial Omics Database (SODB) was established to offer a unified format for data storage and interactive visualization modules. Here we detail the use of Pysodb, a Python-based tool designed to enable the efficient exploration and loading of spatial datasets from SODB within a Python environment. We present seven case studies using Pysodb, detailing the interaction with various computational methods, ensuring reproducibility of experimental data and facilitating the integration of new data and alternative applications in SODB. The approach offers a reference for method developers by outlining label and metadata availability in representative spatial data that can be loaded by Pysodb. The tool is supplemented by a website ( https://protocols-pysodb.readthedocs.io/ ) with detailed information for benchmarking analysis, and allows method developers to focus on computational models by facilitating data processing. This protocol is designed for researchers with limited experience in computational biology. Depending on the dataset complexity, the protocol typically requires ~12 h to complete.
    DOI:  https://doi.org/10.1038/s41596-023-00925-5
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